Cryptosporidium parvum, a protozoan microorganism, is one of principle contributors to water contamination and represents a major threat to human health. Ingestion of just a few oocysts can cause diarrhea and can be especially fatal in immuno-compromised individuals. There is no specific drug therapy proven to be effective to treat cryptosporidial infections. For these reasons, detection of cryptosporidia in water supplies is important. It is also important to be able to distinguish viable and non-viable cryptosporidia and infectious and non-infectious cryptosporidia. 
Cryptosporidia occur outside the body of an animal primarily in the form of oocysts, which are environmentally stable and resistant particles having a diameter that is typically in the range from about 3 to about 6 micrometers. The oocysts are known to remain viable for extended periods of time and are resistant to conventional water disinfection methods. Due to massive shedding of oocysts in the feces of infected animals or individuals and the robust nature of the oocysts, they are frequently present in raw surface water and even in finished drinking water. Each oocyst typically contains four sporozoites, each of which can independently infect a host upon ingestion by the host of the oocyst. Extended exposure to the environment, treatment with certain chemicals, exposure to ultraviolet radiation, and other unknown factors can render sporozoites within an oocyst non-viable, i.e., unable to infect a host upon ingestion of the oocyst.
Current methods used in the water quality testing industry for detection of cryptosporidium oocysts are time-consuming, labor intensive and require highly trained microscopists. These methods rely on microscopic examination of samples that are stained with fluorescent antibodies for the presence of cryptosporidium oocysts. The cross reaction of the antibodies with targets in the sample other than the specific pathogen, often gives false positive results. In the particular case of parasitic protozoa such as cryptosporidium and giardia, if the antibody only reacts with certain variants of the protozoa, but not with the variant present in the water sample being tested, the immunological test can fail to detect the pathogen even when it is present.
In contrast, vibrational spectroscopic techniques such as spontaneous Raman scattering provide specific molecular information on samples. Pathogens can be “fingerprinted” by means of characteristic vibrational frequencies of the molecular species, even in a complex multi-component mixture as disclosed for example in U.S. Pat. No. 6,950,184, which is incorporated herein by reference.
In Raman spectroscopy, incident light having frequency ωp is absorbed by a sample and is re-radiated at a shifted frequency ωs=ωp−Ω, where Ω corresponds to a transition between two vibrational states of molecules in the sample, also referred to as a vibration frequency. The difference between the frequencies of the incident and re-radiated light is known as the Raman shift (RS), and is typically measured in units of wavenumber (inverse length). If the incident light is substantially monochromatic (single wavelength) as it is when using a laser source, the scattered light which differs in frequency can be more easily distinguished by filtering.
As an example, FIG. 2, which is reproduced here from an article by S. Stewart et al, Proc. of SPIE, Vol. 5692, 341-350 (2005), illustrates a typical Raman spectrum of a cryptosporidium oocysts (A) in comparison with a Raman spectrum from river water (B). As seen from the figure, the spontaneous Raman spectrum (A) of cryptosporidium oocysts is dominated by the presence of a strong peak around Raman shift of 2930 cm−1 corresponding to stretching vibrations of a C—H bond, which can be used as an indicator of the presence of cryptosporidium oocysts in water.
One disadvantage of using the aforedescribed spontaneous Raman scattering for water testing relates to low characteristic cross-sections of spontaneous Raman scattering, which resulting in low signal levels and hence considerable amount of time needed to record a Raman spectrum. Additionally, the application of conventional Raman spectroscopy can be disadvantageously affected by a background fluorescence signal, which often limits the sensitivity of detection. Furthermore, the Raman spectra analysis for the detection of cryptosporidium oocysts disclosed in the prior art U.S. Pat. No. 6,950,184 is not capable of discerning between individual organisms and how many oocysts are present in a sample, and is therefore not well suited for quantitative analysis of the oocysts concentration in water.
There is another optical analysis method based on probing vibrational energies of molecules in a sample, namely—a coherent anti-Stokes Raman scattering (CARS) microscopy. CARS is a third order nonlinear optical process and involves simultaneous excitation of a sample under test with two light beams—a pump laser beam at a frequency ωp and a Stokes laser beam at a frequency ωs, resulting in a signal at the anti-Stokes frequency of ωas=2ωp−ωs being generated in a phase matching direction, provided that the frequency difference between the pump and Stokes beams corresponds to a transition between two vibration energy levels of sample molecules, i.e. Ω=ωp−ωs; an energy diagram for this process is shown in FIG. 1. In CARS spectrography, the intensity of the signal at the anti-Stokes frequency ωas is typically plotted as a function of the frequency shift Ω between the pump and Stokes signals and is referred to as the CARS spectrum, with the frequency shift Ω referred to as the anti-Stokes frequency shift or CARS frequency shift and is typically expressed in units of cm−1. Although the CARS microscopy has been applied recently to imaging of live cells in laboratory conditions, see for example U.S. Pat. No. 6,108,081 issued to Holtom et al, it has been largely unknown in the water testing industry.
Therefore the water testing industry currently lacks a method that can provide a fast and reliable detection of water-borne pathogens such as cryptosporidium oocysts and can be used for real-time automated water testing.
An object of the present invention is to overcome the shortcomings of the prior art by providing a method for assessing the presence of individual pathogen organisms in a sample utilizing CARS microscopy for fast pathogen detection and identification.
Another object of the present invention is to provide a method for assessing the presence of individual pathogen organisms in a sample that can be used for automated water monitoring in real-time.